PROJECT SUMMARY Influenza infection increases the incidence of acute MI (AMI) within the first 7 days after detection and the therapeutic benefit of vaccination is comparable to current therapies for secondary prevention of AMI. Platelets are the blood component central to thrombosis and uncontrolled platelet activation is a major contributor to unstable coronary syndromes and AMI. Although thrombotic events have been associated with some viral infections, the molecular mechanisms underlining platelet mediated thrombosis during progression of global viral infection such as influenza have not been established. The major objective of this proposal is to examine the role of a previously undescribed molecular mechanism in platelets that leads to sensing of influenza and ultimately increased thrombosis. The mechanism of reference involves a cytoplasmic receptor known as the retinoic acid-inducible gene-I (RIG-I) receptor and its downstream signaling cascade. Preliminary results show that RIG-I and proteins related to RIG-I molecular signaling become specifically upregulated in platelets from influenza infected patients. Since platelets are anucleated the upregulated expression of RIG-I suggests direct involvement of their precursors the megakaryocytes (MKs) during infection. For that purpose, we will also study the contribution of RIG-I in MKs to platelet reprogramming toward more efficient viral response that may lead to thrombosis. Considering that the RIG-I-signaling axis is present in platelets and specifically upregulated, we hypothesize that during infection, platelets sense influenza through the RIG-I signaling axis leading to their activation and MKs reprogram platelets toward an increased antiviral and thrombotic response. Thus, we propose two specific aims of research: Aim 1. To determine the expression and activity of the RIG-I axis in platelets with respect to their antiviral and/or thrombotic responses; and Aim 2. To characterize how MKs sense influenza and how this sensing subsequently reprograms the RIG-I/MAVS pathway, leading to enhanced expression of antiviral and prothrombotic genes. Building upon novel observations obtained in preliminary studies, our proposed research plan will determine the role of a previously unknown signaling pathway in platelets, RIG-I/MAVS, in mediating influenza antiviral and thrombotic responses. The outcome of this proposal will define the fundamental mechanisms of platelet viral cytosolic signaling, MK response to infection, how this synergistic response contributes to immunity and thrombosis, and how targeting this pathway could ameliorate thrombotic and infectious disease pathologies.